A low-pH-inducible, stationary-phase acid tolerance response in Salmonella typhimurium.

Acid is an important environmental condition encountered by Salmonella typhimurium during its pathogenesis. Our studies have shown that the organism can actively adapt to survive potentially lethal acid exposures by way of at least three possibly overlapping systems. The first is a two-stage system induced in response to low pH by logarithmic-phase cells called the log-phase acid tolerance response (ATR). It involves a major molecular realignment of the cell including the induction of over 40 proteins. The present data reveal that two additional systems of acid resistance occur in stationary-phase cells. One is a pH-dependent system distinct from log-phase ATR called stationary-phase ATR. It was shown to provide a higher level of acid resistance than log-phase ATR but involved the synthesis of fewer proteins. Maximum induction of stationary-phase ATR occurred at pH 4.3. A third system of acid resistance is not induced by low pH but appears to be part of a general stress resistance induced by stationary phase. This last system requires the alternative sigma factor, RpoS. Regulation of log-phase ATR and stationary-phase ATR remains RpoS independent. Although the three systems are for the most part distinct from each other, together they afford maximum acid resistance for S. typhimurium.     

Identification of the cadBA operon from Vibrio vulnificus and its influence on survival to acid stress

By a transposon-tagging method, cadBA genes encoding a lysine/cadaverin antiporter and a lysine decarboxylase were identified and cloned from Vibrio vulnificus. The deduced amino acid sequences of cadBA were 64-97% similar to those reported from other Enterobacteriaceae. Functions of cadBA genes on acid tolerance were assessed by comparing acid tolerances of V. vulnificus and its isogenic mutants, whose cadBA genes were separately inactivated by allelic exchanges. The results demonstrated that gene products of cadBA contribute to acid tolerance of V. vulnificus, and that their contribution is dependent on prior exposure of cells to moderately acidic pH.     

Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine-lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 microM respectively. Both cadaverine uptake and cadaverine-lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine-requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine-lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO(2) and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF-potE operon, encoding inducible ornithine decarboxylase and a putrescine-ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.     

Salmonella acid shock proteins are required for the adaptive acid tolerance response.

Salmonella typhimurium, as well as other enteric bacteria, experiences significant fluctuations in H+ ion concentrations during growth in diverse ecological niches. In fact, some pH conditions which should kill cells rapidly, such as stomach acidity, are nevertheless tolerated. The complete mechanism for this tolerance is unknown. However, I have recently demonstrated that S. typhimurium has the ability to survive extreme low pH (pH 3.0 to 4.0) if first adapted to mild pH (pH 5.5 to 6.0). This phenomenon has been referred to as the acidification tolerance response (ATR). The exposure to mild acid is referred to as preshock, and the proteins involved are called preshock ATR proteins. A second type of encounter with acid, called acid shock, involves shifting cells directly from alkaline conditions (pH 7.7) to acid conditions (pH 4.5 or below). During acid shock, the organism immediately ceases reproduction and dramatically changes the expression of at least 52 proteins. All but four are distinct from the preshock ATR proteins. Surprisingly, acid shock alone did not afford significant protection against strong acid challenge in minimal medium. Furthermore, inhibiting protein synthesis prior to acid shock revealed that the acid shock proteins do not appear to contribute to acid survival in minimal medium even at pH 4.3. Constitutive cellular pH homeostatic mechanisms seem sufficient to protect cells at this pH. The data suggest that the induction of acid shock and preshock ATR proteins are separate processes requiring separate signals. However, for S. typhimurium to survive extreme acid conditions, it must induce both the preshock and acid shock systems. Preventing the expression of one or the other eliminates acid tolerance. I propose a two-stage process that allows S. typhimurium to phase in acid tolerance as the environmental pH becomes progressively more acidic.     

The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs.     

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.     

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.     

The stationary-phase sigma factor σS (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium

The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid. Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor σ^S encoded by rpoS. Differences between the acid responses of virulent S. typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts. The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon σ^S for their synthesis. It is predicted that one or more of these ASPs is essential for the sustained system. The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H₂O₂ and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance. Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance. Consistent with this model, σ^S proved to be induced by acid shock. Our results also revealed a connection between the transient and sustained ATR systems. Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant. One member of the AtbR regulon, designated atrB, was found to be co-regulated by σ^S and AtbR. Both regulators had a negative effect on atrB expression. The results suggest AtrB serves as a link between the sustained and transient ATR systems. When σ^S concentration are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance. Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.     

Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy.

Salmonella typhimurium exhibits a low-pH-inducible acid tolerance response (ATR) that can protect the adapted cell from severe acid challenge (pH 3.3). It is a two-stage system, with some proteins induced at pH 5.8 (pre-acid shock) and others induced below pH 4.5 (acid shock). The genetics of acid resistance was investigated through the use of a new screening medium. The medium contained 200 microM dinitrophenol (DNP) and was adjusted to pH 4.7 to 4.8. The medium will lower the internal pH of cells to a lethal level. However, cells capable of mounting an ATR will survive longer on this medium than acid-intolerant cells. Using this DNP lethal screening strategy, we isolated several acid-sensitive insertion mutants. Some mutants were defective in the pre-acid shock ATR stage but exhibited a normal or nearly normal post-acid shock-induced acid tolerance (atrB and atrC). Others could not induce acid tolerance by using either pre- or post-acid shock strategies (atrD, atrF, and atrG). The atrB locus was found to be part of a regulon under the control of a trans-acting regulator, atbR. An insertion in atbR caused constitutive acid tolerance because of overexpression of the regulon. Mutations in atrD and atrF affected iron metabolism and, in a manner analogous to ferric uptake regulator (fur) mutations, diminished acid resistance. The atrF mutation mapped within the ent cluster, probably in a fep uptake locus. The atrD locus mapped near metC and may represent an insertion into the S. typhimurium homolog of the Escherichia coli exbB or exbD locus. The mutation in atrC caused extreme UV light sensitivity and proved to occur within the polA (DNA polymerase I) locus. The results support the concept of overlapping acid protection systems in S. typhimurium.     

Adaptive acidification tolerance response of Salmonella typhimurium.

Salmonella typhimurium can encounter a wide variety of environments during its life cycle. One component of the environment which will fluctuate widely is pH. In nature, S. typhimurium can experience and survive dramatic acid stresses that occur in diverse ecological niches ranging from pond water to phagolysosomes. However, in vitro the organism is very sensitive to acid. To provide an explanation for how this organism survives acid in natural environments, the adaptive ability of S. typhimurium to become acid tolerant was tested. Logarithmically grown cells (pH 7.6) shifted to mild acid (pH 5.8) for one doubling as an adaptive procedure were 100 to 1,000 times more resistant to subsequent strong acid challenge (pH 3.3) than were unadapted cells shifted directly from pH 7.6 to 3.3. This acidification tolerance response required protein synthesis and appears to be a specific defense mechanism for acid. No cross protection was noted for hydrogen peroxide, SOS, or heat shock. Two-dimensional polyacrylamide gel electrophoretic analysis of acid-regulated polypeptides revealed 18 proteins with altered expression, 6 of which were repressed while 12 were induced by mild acid shifts. An avirulent phoP mutant was 1,000-fold more sensitive to acid than its virulent phoP+ parent, suggesting a correlation between acid tolerance and virulence. The Mg2(+)-dependent proton-translocating ATPase was also found to play an important role in acid tolerance. Mutants (unc) lacking this activity were unable to mount an acid tolerance response and were extremely acid sensitive. In contrast to these acid-sensitive mutants, a constitutively acid-tolerant mutant (atr) was isolated from wild-type LT2 after prolonged acid exposure. This mutant overexpressed several acidification tolerance response polypeptides. The data presented reveal an important acidification defense modulon with broad significance toward survival in biologically hostile environments.